Despite continuous efforts to find new effective therapies, heart failure (HF) remains one of the most common causes of morbidity and mortality in Western societies. One approach that has been pursued in recent years is the identification of biomarkers to aid in the classification, diagnosis and prognosis of heart failure and other cardiac conditions. In particular, the presence and particular peptides and proteins have been found to be useful diagnostic and prognostic indicators. For example, the phosphorylation of desmin at novel sites has been found to be indicative of an increased risk of heart failure in a patient, as disclosed in International Application No. PCT/US2010/036228, which is incorporated herein by reference.
Investigating individual phosphorylation residues is challenging but essential for understanding cellular signaling and the effect on protein function. To unravel these phosphorylation-dependent structure-function relationships, a quantitative analysis of residue-specific protein phosphorylation and the presence or absence of the N- or C-terminus is required. Phosphorylation of myofilament proteins has been found to play a pivotal role in regulating cardiac contraction. Current approaches to characterize the phosphorylation status are limiting, only able to distinguish one or two different phosphorylated residues within a protein. These techniques rarely allow for a simultaneous, global view of multiple post-translational modified forms (e.g. phosphorylated residues) in a given protein, nor can they provide quantitative information about phosphorylation levels at each residue. Overcoming these limitations will be of major importance for the analysis of a clinically relevant protein such as human cardiac Troponin I (cTnI), a clinical indicator of myocardial damage.
Release of cardiac proteins in the blood, and specifically immunoassay of cTnI in serum is one of the gold standards for diagnosis of acute myocardial infarction. However these diagnostic assays do not specifically assay modifications of the protein which we have associated with heart failure as described in this application. In the heart, cTnI plays a regulatory and inhibitory role as part of the thin filament. It function is regulated by phosphorylation. There are six residues that are known to be phosphorylated by several kinase (Ser23/24: target by PKA, PKC, PKD, PKG; Ser42/44, Thr143: target by PKC; Ser150: target by p21-activated kinase (PAK) [1]. The alterations of phosphorylation level on these residues can have dramatic effects on cardiac function and are involved in various cardiac diseases such as cardiac hypertrophy and heart failure (2-4). However, the identification and quantification of multiple phosphorylated residues within the degraded or intact cTnI by immunoassay- or traditional mass spectrometry (MS)-based approach has proven challenging because of the lack of specific antibody to nearly all sites (only PKA and PKC sites are available and they are not to individual sites), high costs along with a long time to develop new specific antibodies, substoichiometric phosphorylation, facile loss of phosphoric acid, variable ionization efficiency with the changing modification status, and the intrinsic molecular complexity of human cTnI. As a result, the basal phosphorylation status of human cTnI has not been adequately established in vivo. Even less is known about the phosphorylation status of cTnI in failing conditions such as Ischemic heart disease (ISHD) and Idiopathic dilated cardiomyopathy (IDCM). To overcome this limitation, our laboratory has developed a MS-based target proteomics quantification method—Multiple Reaction Monitoring (MRM) assay (for more information about MRM see our recent review [5]) for both targeted and global analysis of cTnI, which allows us to systematically identify and quantify the extent of specific post-translational modification including at each potentially phosphorylated residue.
This application claims the benefit of the filing date of provisional patent application no. 61/305,298, filed Feb. 17, 2010, which is incorporated by reference in its entirety herein.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.